DESCRIPTION (Adapted from abstract): Study of the streptavidin- biotin model system is providing molecular insight into the structure-function relationships, which govern the high affinity equilibrium thermodynamics and the construction of the high activation barrier to dissociation. A mechanistic model of the dissociation reaction coordinate is also emerging and further studies will test the validity of the transition state structural model by both experimental and computational techniques. Because streptavidin utilizes common aromatic, hydrogen bonding, and flexible loop interaction motifs to generate uncommonly high- affinity, the principles elucidated here should prove generally useful to the field of structure-based drug design. In order to connect fundamental advances to drug design, the investigators have added a computational component. State-of-the-art computational approaches will help interpret the thermodynamic findings, investigate the ligand exit pathway, and connect our findings to structure based drug design algorithms. Two new questions concerning the roles of particular protein residues in ligand binding are the focus of this project. First, are some side-chain contacts designed to manage the enthalpic or entropic components of binding free energy depending on the specific physical properties of the portion of the ligand they interact with? For example, do residues contacting portions of a ligand expected to have high configurational entropy (such as the valeric acid tails of biotin) have a different energetic challenge than those residues contacting portions of the ligand which are relatively rigid (such as the bicyclic ring portion of biotin)? The second question is whether particular amino acids manage activation barrier energetics rather than primarily serving as equilibrium contacts? In enzymes, some side-chains are designed to preferentially manage the activation barrier. Is that true for some protein-ligand contacts as well? This project will fill important gaps in the fundamental understanding of 1) what is called mechanistic thermodynamics - which describes how aromatic, hydrogen bonding, and flexible loop side-chain contacts manage the enthalpic and entropic costs of ligand immobilization, 2) the construction of ligand dissociation activation barriers and how binding contacts manage activation enthalpies and entropies, and 3) ligand dissociation mechanisms and whether there are defined ligand exit pathways. This project could also provide useful input for the design of enzymes (e.g., catalytic antibodies), where the problem of product inhibition is essentially a problem of the dissociation activation barrier. The streptavidin mutants could also be useful reagents in affinity separations, diagnostics, and targeted drug discovery.